Today, plant biotechnology relies on two approaches for delivery and expression of foreign genes in plants: stable genetic transformation and transient expression. The last one can be built on agro-infiltration or viral infection (for review see: Fischer et al., 1999, Biotechnol. Appl. Biochem., 30, 113-116). Transient expression can be achieved by agroinfiltrating plant tissue with a standard expression cassette under control of a constitutive, for example 35S promoter to drive expression of gene of interest (Vaquero et al., 1999, Proc. Natl. Acad. Sci. USA., 96, 11128-11133) or, at larger scale, by transfecting a plant with viral vectors. Usually agro-infiltration does not provide for high yield, but in combination with post-transcriptional gene silencing (PTGS) suppressors, like p19 or HcPro the protein expression level can be increased up to 50-folds (Voinnet et al., 2003, Plant J., 33, 549-556). Still it is far below the biological limits that can be achieved with the help of viral expression systems (Marillonnet et al., 2004, Proc. Natl. Acad. Sci. USA, 101, 6852-6857). A description of a bioreactor based on the use of agro-infiltration for transient expression of a recombinant protein of interest in plant tissue is provided in U.S. Pat. No. 6,740,526. However, this patent is silent on how to improve the yield of the protein to be expressed, i.e. it does not go beyond other known processes in the field (Vaquero et al., 1999, Proc. Natl. Acad. Sci. USA., 96, 11128-11133). A more powerful approach for transient expression is the use of viral vectors. It was shown that TMV-based expression of a reporter gene (GFP or DsRed) can reach the biological yield limits of the system, producing several milligrams of recombinant protein per gram of fresh leaf biomass (Marillonnet et al., 2004, Proc. Natl. Acad. Sci. USA, 101, 6852-6857). The relative yield in such system can reach up to 80% of TSP, thus significantly facilitating and reducing the cost of downstream processing. Such a high relative yield is possible due to virus-induced shut-off of host protein biosynthesis.
It is evident that viral vectors can provide for higher expression levels than conventional vectors used in agro-infiltration (for review see: Porta & Lomonossoff, 1996, Mol Biotechnol., 5, 209-221; Yusibov et al., 1999, Curr. Top. Microbiol. Immunol., 240, 81-94) and are a powerful tool for functional genomics studies (Dalmay et al., 2000, Plant Cell, 12, 369-379; Ratcliff et al., 2001, Plant J., 25, 237-245; Escobar et al., 2003, Plant Cell, 15, 1507-1523). Numerous publications and patents in the field describe systems based on DNA and RNA viral vectors (Kumagai et al., 1994, Proc. Natl. Acad. Sci. USA, 90, 427-430; Mallory et al., 2002, Nature Biotechnol. 20, 622-625; Mor et al., 2003, Biotechnol. Bioeng., 81; 430-437; U.S. Pat. No. 5,316,931; U.S. Pat. No. 5,589,367; U.S. Pat. No. 5,866,785; U.S. Pat. No. 5,491,076; U.S. Pat. No. 5,977,438; U.S. Pat. No. 5,981,236; WO02088369; WO02097080; WO9854342). The existing viral vector systems are usually restricted to a narrow host range in terms of their best performance and even the expression level of such vectors in their most favourable host is far below the upper biological limits of the system. An important issue of virus-based systems is the method of delivery of the viral replicon to a plant cell. The most broadly applied method of delivery for large-scale production (simultaneous production in many plants, e.g. in a farm field or a greenhouse) is the use of infectious copies of RNA viral vectors (Kumagai et al., 1995, Proc. Natl. Acad. Sci. USA, 92, 1679-1683). Because of a relatively high tendency of recombinant viral RNA vectors to lose the heterologous inserts during cycles of their replication, the method requires transcription of DNA templates in vitro, and is therefore inefficient and expensive.
Although much faster, the transient route is very limited because of the virus's low infectivity, inability to transfect most of the plant body, and gene size limitations. There are publications describing the use of agro-infiltration for delivery of infectious viral vectors into the plant cell (Liu & Lomonossoff, 2002, J. Virol. Methods, 105, 343-348) or assembly of viral vectors from agrobacterium-delivered viral vector components (Marillonnet et al., 2004, Proc. Natl. Acad. Sci. USA, 101, 6852-6857). However, these publications do not address the issue of efficient and synchronized formation of viral replicons in each plant cell of agro-infiltrated tissue and further vector spread depends on the ability of the virus for cell-to-cell and systemic movement. This movement requires a relatively long time and usually viral vectors provide for the highest possible yield for said vector in approx. 10-14 days after infection. This is not acceptable for the production of recombinant proteins that strongly interfere with plant cellular processes, especially highly cytotoxic proteins like restriction enzymes, proteases, non-specific nucleases, many pharmaceutical proteins. In contrast, a standard vector delivered via agro-infiltration reaches its highest possible expression level in 3-4 days after agro-delivery, but the yield provided by such a vector is unacceptably low. We therefore face the problem caused by the low efficiency of standard transcriptional vectors driven by constitutive promoters to express the protein of interest, despite these vectors provide for expression of gene(s) of interest in practically every cell of agro-infiltrated plant tissue, and vice-versa, agro-delivered viral vectors are capable of providing for high expression level, but rarely initiate replication and thus do not provide for expression in the majority of cells, but only in a small fraction (less than 1% of all agroinfiltrated tissue). As the result, such virus-based vectors require significantly (3-4 folds) longer time to provide for expression, but productivity of the system remains lower than it could theoretically be, especially in case of cytotoxic proteins. This is serious drawback for using viral vectors in transient expression systems, as they do not provide for synchronized expression in agro-infiltrated tissue, thus affecting the yield, especially in case of cytotoxic genes to be expressed. Also, an infected plant host does not contain viral vectors in a large proportion of its tissues, thus excluding tissues from the production process. Additionally, the time required for achieving the best possible spreading (and expression level) of a viral vector over the infected plant is 3-4 times longer in comparison with standard agro-infiltration protocols. Moreover, none of the described systems for transient expression addressed the issue of increasing the biological safety of the system, which is an important element in industrial scale protein production involving the use of genetically engineered organisms.
Despite many publications in the field including patented technologies, there are still no large scale virus-based production systems that work with sufficient efficiency and yield for commercial high-yield production, predominantly due to two main reasons:
Firstly, transient plant virus-based expression systems are generally restricted to specific hosts, which may not be suitable for large scale cultivation due to their susceptibility to environmental factors. Moreover, they are generally restricted to certain parts of a plant host, thus excluding most of the plant biomass from the production process and as a result minimizes the relative yield of recombinant product per unit of plant biomass down to a level comparable to that achievable by a conventional transcription promoter in a transgenic plant;Secondly, attempts to scale up the virus-based production system by generating transgenic plant hosts having the viral replicon precursor stably integrated in each cell has not provided a solution either, in particular because of underperformance of said replicons in such position, “leakiness” of the gene of interest to be expressed from said replicon and lack of an efficient switch system for said vectors. Some progress was achieved with PVX-based vectors by using suppressors of PTGS silencing as trigger of RNA replicon formation (Mallory et al., 2002, Nature Biotechnol., 20, 622-625), but the system is still far below the practical value, as there is no solution provided for an efficient control of the switch (PTGS suppressor) triggering viral vector replication. However, this system provided for an expression level of the GUS gene reaching 3% of total soluble protein (TSP), which is the best known so far for this type of system, but still no better than a conventional transgene expression system under control of a strong promoter. Another inducible system based on a plant tripartite RNA virus (Mori et al., 2001, Plant J., 27, 79-86), Brome Mosaic Virus (BMV), gave a very low yield of the protein of interest (3-4 μg/g fresh weight), which is comparable with the yields provided by standard transcriptional promoters.
The low expression levels achieved so far with plant expression systems are a major reason why these systems are hardly competitive with other expression systems like bacterial, fungal, or insect cell expression systems. Low expression levels give rise to very high downstream costs for protein isolation and purification in a huge background of plant material. Therefore, costs for downstream processing quickly decrease, as the yield of the protein or product of interest per unit plant biomass increases.
There is presently no large-scale plant transient expression system the yield and efficiency of which would be sufficiently high to compete on the market with other large-scale expression systems like bacterial, fungal, or insect cell expression systems. Such a plant expression would have to fulfill the following criteria as good as possible:    (i) high yield, including expression of the protein of interest in as many plant tissues as possible and in as many cells of said tissues;    (ii) for preventing a deleterious effect of protein expression on plant cells survival, expression of the protein or product of interest should start in all plant cells of the treated plant or plant tissue at the same time.            Typically, the protein or product of interest accumulates in each cell producing said product or protein up to a certain point. During accumulation, however, degradative processes frequently set on that tend to reduce the yield or quality of the protein or product of interest. Therefore, there is an optimal point in time after switching on expression, where the product or protein of interest should be harvested. This optimal point in time should be reached in all tissues or cells of a plant and in all plants of a selected lot at the same time in order to make the overall process efficient and profitable;            (iii) the system shall incorporate increased biosafety features, such that agrobacteria used for agroinfiltration shall have at least one of the following features: low or zero survival rate in open environment, low or zero infectivity toward non-target organisms or non-target plants.Therefore, it is an object of the invention to provide a process for protein expression in a plant system that is scalable to large-scale applications, gives a high yield of the protein to be expressed, and, at the same time, is biologically safe in that the probability of transfection or transformation of non-target organisms with foreign DNA is low.